Vaccines have been very effective in protecting people from a wide variety of diseases, whether caused by viruses, bacteria, or fungi. The ability of vaccines to induce specific protection against such a wide range of pathogenic organisms results from their ability to stimulate specific humoral antibody responses, as well as cell-mediated responses. This invention relates to a process for preparing such vaccines, and particularly to a process for making conjugates that are used in preparing vaccines. Additionally, the process of the invention can be used to produce immunogens and other valuable immunological, therapeutic, or diagnostic reagents. The invention further relates to the vaccines, immunogens, and reagents produced from the conjugates made according to the invention, as well as to the use of these products.
It is often very desirable to induce immune responses against polysaccharides. For example, antibodies against a bacterial capsular polysaccharide can provide protection against that bacterium. Many polysaccharides, however, are poorly immunogenic, particularly in infants and young children. Furthermore, in both children and adults, there is usually no booster effect with repeated polysaccharide immunizations, and the principal antibody class is IgM. These features are all characteristic of so called "T cell independent" ("TI") antigens.
In many cases, the immunogenicity of polysaccharides can be enhanced by covalently linking proteins or T cell epitope-containing peptides to the polysaccharide. Certain other components, such as lipids, fatty acids, lipopolysaccharides, and lipoproteins, also are known to enhance the immunogenicity of the polysaccharide. As described in the "dual conjugate" patent application of Mond and Lees, conjugation of a protein to a polysaccharide can enhance the immune response to the protein as well as to the polysaccharide. See U.S. Pat. No. 5,585,100; U.S. patent application Ser. No. 08/444,727 (filed May 19, 1995); and U.S. patent application Ser. No. 08/468,060 (filed Jun. 6, 1995). These patent applications each are entirely incorporated herein by reference. This effect also is described in A. Lees, et al., "Enhanced Immunogenicity of Protein-Dextran Conjugates: I. Rapid Stimulation of Enhanced Antibody Responses to Poorly Immunogenic Molecules," Vaccine, Vol. 12, No. 13, (1994), pp. 1160-1166. This article is entirely incorporated herein by reference. In view of this potential for improving the immune response against polysaccharides, there is a need in the art for methods to covalently link proteins or other moieties to polysaccharides.
Ideally, the process of covalently linking moieties to a polysaccharide must be done in a way to maintain antigenicity of both the polysaccharide and protein components and to minimize damage to necessary epitopes of each component. Furthermore, the linkage should be stable. Therefore, there is a need for a mild and gentle means for coupling proteins, peptides, haptens, organic molecules, or other moieties to polysaccharides.
Vaccines are not the only products that can benefit from an improved procedure for coupling molecules together. For example, certain diagnostic or therapeutic reagents are produced by coupling polysaccharides, high molecular weight carbohydrates, and low molecular weight carbohydrates to solid phase materials (e.g., solid particles or surfaces). Thus, there is a need in the art for improved means for coupling polysaccharides, high molecular weight carbohydrates, and low molecular weight carbohydrates to solid phase materials.
Two main methods for coupling molecules together are used. In the first method, the means for coupling entails the crosslinking of a protein (or peptide or other moiety) directly to a polysaccharide (or some other moiety). Sometimes, however, a spacer molecule is needed between the coupled moieties, either to facilitate the chemical process and/or to enhance the immune response to the protein and/or the polysaccharide. In either method, it is usually necessary to activate or functionalize the polysaccharide before crosslinking occurs. Some methods of activating or functionalizing polysaccharides are described in W. E. Dick, et al., "Glycoconjugates of Bacterial Carbohydrate Antigens: A Survey and Consideration of Design and Preparation Factors," Conjugate Vaccines (Eds. Cruse, et al.), Karger, Basel, 1989, Vol. 10, pp. 48-114. This excerpt is entirely incorporated herein by reference. Additional activation methods are described in R. W. Ellis, et al. (Editors), Development and Clinical Uses of Haemophilus B Conjugate Vaccines, Marcel Dekker, New York (1994), which book is entirely incorporated herein by reference.
One preferred method for activating polysaccharides is described in the CDAP patent applications of Lees, U.S. patent application Ser. No. 08/124,491 (filed Sep. 22, 1993, now abandoned); U.S. Pat. Nos. 5,651,971; 5,693,326; and U.S. patent application Ser. No. 08/482,666 (filed Jun. 7, 1995). These U.S. patents and patent applications each are entirely incorporated herein by reference. The use of CDAP also is described in Lees, et al., "Activation of Soluble Polysaccharides with 1-Cyano-4-Dimethylamino Pyridinium Tetrafluoroborate for Use in Protein-Polysaccharide Conjugate Vaccines and Immunological Reagents," Vaccine, Vol. 14, No. 3 (1996), pp. 190-198. This article also is entirely incorporated herein by reference.
One specific method of preparing conjugates is through the condensation of amines (or hydrazides) and carboxyls to amides using carbodiimides. The carboxyl nucleophile reacts with the carbodiimide to form a highly reactive but unstable intermediate that can then either hydrolyze or react with an amine to form a stable amide bond. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide ("EDC") is a water soluble example of this class of carbodiimide reagent.
As one example of this reaction, Robbins describes functionalizing Haemophilus influenza ("PRP") polysaccharide with hydrazides and condensing this functionalized material with carboxyls on tetanus toxoid. See C. Chu, et al., Infection and Immunity, Vol. 40, 1983, beginning at pg. 245. Additionally, the coupling of a carboxylated polysaccharide to diptheria toxoid by this general process also is described by Robbins. See S. C. Szu, et al., Journal of Experimental Medicine, Vol. 166, 1987, beginning at page 1510. These articles each are entirely incorporated herein by reference.
In general, however, there are a myriad of problems when one attempts to use carbodiimide for coupling multivalent ligands (e.g., proteins and polysaccharides) that contain both activatable groups and nucleophiles. The reaction is difficult to control, and it frequently leads to extensive homopolymerization, interchain crosslinking, and reduced antigenicity. A further problem is that the carboxyl-carbodiimide intermediate can undergo an O to N acyl shift, resulting in a stable, unreactive addition product that adds new epitopes to the protein (see G. T. Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif., (1996), which document is entirely incorporated herein by reference).
Another method of forming conjugates is through the use of active ester intermediates. Reagents that form active ester intermediates include norborane, p-nitrobenzoic acid, NHS (N-hydroxysuccinimide), and S-NHS (sulfo-N-hydroxysuccinimide). NHS esters (or other suitable reagents) can react with nucleophiles like amines, hydrazides, and thiols. The reaction products of NHS esters with amines and hydrazides are particularly stable, forming an amide bond. NHS ester intermediates can be formed in a one step process using carbodiimide (to activate the carboxyls) and NHS (or S-NHS). In this process, NHS (or S-NHS), the carboxyl-containing component, and the amine-containing component are combined, and the carbodiimide is added thereto. Although coupling efficiency often is higher in this reaction than is the case when NHS is not present, problems, such as homopolymerization, interchain crosslinking, and over-crosslinking, can occur in this process. Additionally, other undesirable side reactions can occur and cause problems, as will be described in more detail below.
Alternatively, a two step activation process can be used. In this procedure, one attempts to remove or destroy the remaining carbodiimide before adding the component to be crosslinked. In one protocol using EDC and NHS, before adding the protein, the remaining carbodiimide is deactivated with a thiol (e.g., mercaptoethanol). See Grabarek and Gergely, Analytical Biochemistry, Vol. 185 (1990), beginning at pg. 131, which article is entirely incorporated herein by reference. By this method, the amount of carbodiimide present during protein addition is minimized. The addition of the thiol, however, also can hydrolyze the desired NHS ester intermediate. In this two step process, it would be preferable to isolate the NHS ester intermediate. It can be difficult, however, to isolate this intermediate because it is only moderately stable in aqueous media.
An additional problem with carbodiimide/NHS procedures is the possible formation of a .beta.-alanine derivative resulting from the reaction of carbodiimide with two moles of NHS in a Lossen rearrangement (see Wilchek and Miron, Biochemistry, Vol. 26, beginning at pg. 2155, 1987, which article is entirely incorporated herein by reference). This derivative can react with amines to form an unstable crosslink.
Carbodiimide and NHS also have been used to activate oligosaccharides. In such procedures, the reducing ends of oligosaccharides are functionalized with carboxyl groups and then converted to active esters using carbodiimide and NHS in organic solvents. These functionalized oligosaccharides are then coupled to proteins. See Porro, U.S. Pat. No. 5,153,312 (Oct. 6, 1992) for the use of this procedure with an oligosaccharide from Neisseria meningiditis polysaccharide type C. This patent is entirely incorporated herein by reference. The reported overall coupling efficiency, however, is low, and low molecular weight oligosaccharides are used. One reason for the low coupling efficiency is that the oligosaccharides have only one NHS per molecule. It either hydrolyzes or couples.
A variety of other reagents are known for introducing NHS esters; however, most of these require dry organic solvents and are unsuitable for use in aqueous media. One exception includes certain uronium salts, such as the reagent O-(N-succinimidyl) N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU), which are somewhat stable in water, although more so in mixed organic/aqueous media. TSTU has been used to form NHS esters of low molecular weight molecules in organic solvents (see Moller et al., "Versatile Procedure of Multiple Introduction of 8-Aminomethylene Blue into Oligonucleotides," Bioconjugate Chemistry, Vol. 6 (1995), pp. 174-178; Lefevre et al., "Texas Red-X and Rhodarnine Red-X, New Derivatives of Sulforhodamine 101 and Lissamine Rhodamine B with Improved Labeling and Fluorescence Properties," Bioconjugate Chemistry, Vol. 7 (1996), pp. 482-489; and Bannwarth et al., "219, Bathophenanthroline-ruthenium (II) Complexes as Non-Radioactive Labels for Oligonucleotides which can be Measured by Time-Resolved Fluorescence Techniques," Helvetica Chimica Acta, Vol. 71 (1988), beginning at pg. 2085, which articles each are entirely incorporated herein by reference.) Additionally, TSTU and other uronium salts have been used to form NHS esters of low molecular weight molecules in mixed organic/aqueous media (see Knorr et al.,"New Coupling Reagents in Peptide Chemistry," Tetrahedron Letters, Vol. 30, No. 15 (1989), pp. 1927-1930; and Bannwarth and Knorr, "Formation of Carboxamides with N,N,N',N'-Tetramethyl (Succinimido) Uroniumn Tetrafluoroborate in Aqueous/Organic Solvent-Systems," Tetrahedron Letters, Vol. 32, No. 9 (1991), pp. 1157-1160, which articles also are entirely incorporated herein by reference).
TSTU also has been used to prepare active esters of solid phase carboxylated beads in organic solvents (see Wilchek et al., "Improved Method for Preparing N-Hydroxysuccinimide Ester-Containing Polymers for Affinity Chromatography," Bioconjugate Chemistry, Vol. 5 (1994), pp. 491-492, which article is entirely incorporated herein by reference). Reagents like TSTU are advantageous over the carbodiimide/NHS method because there is a reduced likelihood of various side reactions, such as an O to N shift reaction or a Lossen rearrangement.
M. A. Andersson, et al., "Synthesis of oligosaccharides with oligoethylene glycol spacers and their conversion into glycoconjugates using N,N,N',N'-tetramethyl(succinimido)uronium tetrafluoroborate as a coupling reagent," Glycoconjugate Journal, Vol. 10 (1993), pp. 461-465, which article is entirely incorporated herein by reference, describes the use of TSTU to activate a carboxylated saccharide in a mixed aqueous/organic solvent and the subsequent coupling of this activated material to a protein. Andersson does not describe the use of this method for producing vaccines.
European Patent Application No. 0,569,086 A2 (S. J. Danielson et al.) describes the use of TSTU and similar reagents for preparing active esters of insoluble carboxylated substrates and particles. These activated solids are subsequently coupled to biologically relevant molecules to prepare diagnostic reagents. This document is entirely incorporated herein by reference.
Despite the various coupling and activation methods described in the various documents mentioned above, there is an on-going need in the art for improved methods for coupling biologically relevant molecules to one another to produce vaccines. Additionally, there is a need in the art for an improved procedure for coupling biologically relevant molecules to non-carboxylated surfaces and particles to produce various reagents. This invention seeks to provide an improved coupling method for producing conjugates for vaccines and immunogens. In addition, these methods will be useful for producing immunological reagents, diagnostic reagents, and therapeutic reagents.